| Objective: Neural crest stem cells(NCSCs)is a transient and pluripotent cell group that occurs in the development of vertebrate embryos.One of its characteristics is the long distance migration from neural crest to the goal organs in a specific pathway.However,the pathway and regularity of NCSCs migration are not yet clear.In this study,the pathway and regularity of NCSCs migration were observed through immunohistochemical staining,the CXCR4 expression in NCSCs as well as the trend of SDF-1 along NCSCs migration was also carried out by immunofluorescence staining,which preliminarily explored the role of SDF-1/CXCR4 axis in driving NCSCs migration.Methods: 1.Specimen preparation: the whole rat embryos were obtained in E9,E9.5,E10,E10.5,E11,E12,E13 and E14,followed by fixed,routine dehydrated,paraffin embed and continuous sliced into 3.5?m,the slices were prepared for the next staining.2.Immunohistochemical staining: slices in E10 and E12 were used for RET,p75 NTR,Pax7 and Sox10 immunohistochemical staining to screen the candidate markers of NCSCs.Slices in E9,E9.5,E10,E10.5,E11,E12,E13 and E14 were used for Sox10 immunohistochemical staining to observe the pathway and regularity of NCSCs migration.3.Immunofluorescence staining: Sox10 and CXCR4,Sox10 and SDF-1/CXCR4 were double immunofluorescence stained to observe the sequential expression of CXCR4 and SDF-1 in NCSCs migration with Sox10 as marker.Results:1.Selection of candidate markers of NCSCs: Immunohistochemical staining of Sox10,Pax7,P75 NTR and RET in NCSCs were carried out in different developmental stages(E10 and E12).The results showed that in both E10 and E12,Sox10 was the most obvious;the sensitivity and specificity were both strong indicating Sox10 was the preferred markers of NCSCs.2.The pathway and regularity of NCSCs migration: By Sox10 immunohistochemical staining,the migration pathway of NCSCs in rat embryos was demonstrated.The results showed that neural crest began to form and to produce NCSCs in E9.5,NCSCs migrated symmetrically in both sides along the gap between neural tube and epidermis.NCSCs migrated into the thyroid gland and both sides of the chordae in E10-10.5.In E11,NCSCs reached the dorsal aorta,partially into the bilateral surround heart cavity.In E12,NCSCs colonized the pericardial;a part of cells began to enter the anterior intestinal,middle intestine,and pancreas spleen through the original diaphragm.NCSCs began to colonize the hintgut and differentiate in E13,and finally ENS is formed at E14.3.Sequential expression of SDF-1/CXCR4 in NCSCS migration: CXCR4 was expressed in cell membranes and cytoplasm of NCSCs.NCSCs always migrates along the SDF-1 concentration gradient.Sox10-positive NCSCs and SDF-1 have a high degree of orientation.Conclusion:1.Sox10 is the preferred marker for NCSCs in the immunological staining of paraffin section to RET,p75 ntr and Pax7.2.On the beginning of E9.5,neural crest is the first to form and produce NCSCs;NCSCs migrate symmetrically to both sides along the gap between neural tube and epidermis.In E10-10.5,NCSCs migratesn to the thyroid gland and both sides of the chordate;in E11,NCSCs reached the dorsal aorta on both sides,partially into the bilateral surround heart cavity;in E12,NCSCs enters in the pericardial colonization,a part of cells begin to enter the anterior intestinal,middle intestine and pancreas spleen through the original diaphragm;in E13,NCSCs began to colonize in the intestinal and to differentiate,ENS is formed at E14.3.CXCR4 was expressed in cell membranes and cytoplasm of NCSCs.ENCSCs always migrate along the SDF-1 concentration gradient;SDF-1/CXCR4 axis plays an important role in NCSCs migration. |
PDF Full Text Request